Carcinogenesis is a multi-step process involving the activation of cellular proto-oncogenes and inactivation of tumor suppressor genes. Epithelial cells undergo different stages of phenotypic and genotypic alterations and gradually acquire malignant growth characteristics. In spite of the relatively high frequency of oral cancers, little is known about the molecular events that lead to disease initiation and progression. The goal of our study is to develop an in vitro model of oral carcinogenesis using immortalized human gingival keratinocytes (IHGK) in culture and to characterize the growth and differentiation properties of normal human gingival keratinocytes (NHGK) IHGK and oncogene-transfected IHGK cells. We also compared the properties of several head and neck cancer cell lines (HN cells) with NHGK and IHGK in an effort to find biomarkers for oral cancer.Growth and terminal differentiation of NHGKWe prepared primary cultures of NHGK from healthy tissue of the retromolar pad by separation of epithelium by dispase, followed by trypsin digestion. In KGM medium, optimum growth and plating efficiency of oral keratinocytes were observed at 0.15 mM Ca++, whereas the optimum Ca++ concentration for human skin keratinocytes is 0.05 mM. When NHGK cells reached post-confluency in KGM media containing 0.15 - 1.2 mM Ca++, induction of TGase I activity (by 5-10-fold) was followed by formation of insoluble cell envelopes (CEs) crosslinked by TGase I. This finding suggests that NGHK cells in culture. Like skin keratinocytes, can be induced to undergo terminal differentiation in vitro. RT-PCR and Western blot analysis also showed evidence for an increase in TGase I mRNA and protein, suggesting that TGase I is the key marker for terminal differentiation of oral keratinocytes. The mRNA levels of other markers of terminal differentiation, e.g. involucrin, SPR1 and annexin I, were also increased in the Ca++-induced NHGK cells. The amino acid composition of the cross-linked cornified envelope, shows a very high level of proline (12-18%), suggesting that the small proline-rich protein (SPR) is a major component of cornified envelopes of NHGK. When these cornified envelopes were digested with protease, trypsin or chymotrypsin, a number of peptides with amino acid sequences corresponding to those of SPR1, annexin I, loricrin, envoplakin, desmoplakin, involucrin, cystatin alpha and pancornulin were identified. The amino acid composition and protein composition of NHGK cornified envelopes are clearly different from those of normal epidermal keratinocytes (NHEK) cornified envelopes suggesting a specialized barrier function of oral epithelium distinct from that of the skin.Expression of TGase I and TGase II in HN SCC cellsTGase I, a keratinocyte-specific transglutaminase is intimately involved in terminal differentiation. The cellular function of TGase II is less well defined, although it has been implicated in apoptosis, cell-matrix interactions, metastasis and signal transduction. We have compared the expression of cytokeratins 1, 5, 8, 10, 14 and 19, TGase I and TGase II in NHOK cells and several head and neck cancer cells (HN4, HN12, HN8, HN22, HN30 and HN31). The expression of the major cytokeratins (1, 5, 10 and 14) was significantly reduced in most head and neck SCCs. Aberrant expression of cytokeratin 8 or 19 was observed in some carcinoma cells. The level of TGase I was very low (~10% of NHGK) in all oral SCC cell lines and was not induced in post-confluent culture, consistent with their lost ability for terminal differentiation. TGase II was not inducible but a drastic increase in TGase II was observed in HN12, HN3 12:00 AM and HN31 cells. The level of TGase II activity of the HN cells appeared to correlate with their ability to grow in soft agar, suggesting a role for T Gase II in anchorage-independent growth. TGase II activity, however, did not correlate with the tumorigenicity of these cells. Immortalization and oncogene transfection. Since primary keratinocytes have a limited life span in culture (~40 generations), immortalization is a prerequisite step to any long-term experiments. We have used a pLXSN vector containing HPV16 E6/E7 genes and a neomycin resistance marker, or a pBabe vector containing HPV16 E6/E7 genes and a hygromycin resistance marker for immortalization of NHGK. After transfection with these vectors and selection, immortalized gingival keratinocyte cell lines were derived. The immortalized human gingival keratinocytes (IHGK) cells show a similar pattern of cytokeratin expression as does NHGK. Like NHGK cells, IHGK cells undergo terminal differentiation upon reaching post-confluency in a high Ca++ medium. However, the capacity of IHGK to induce TGase I and to form insoluble cornified envelopes seems decrease with the increased number of passages in culture. To the hygromycin-resistant IHGK cells various human oncogenes, including N-ras, K-ras, c-Raf, c-myc, the viral oncogene MSV-H-ras and its activated form, MSV-H- ras-V12, were introduced using the pCEFL vector, and G418- resistant cells were isolated. We are characterizing the growth properties of these oncogene-transfected cells in liquid and in soft agar, their Ca++ resistance, and their ability to form tumors in nude mice.